The heterotrimeric G proteins, a family of GTPases, are present in all cells. They control a variety of functions (metabolic, humoral, neural and developmental) by transducing hormonal, neurotransmitter and sensory signals into an array of cellular responses. Triggered by cell surface receptors, each G protein regulates the activity of a specific effector. The effectors include adenylate cyclase, phospholipase C, and ion channel proteins which initiate appropriate biochemical responses. G proteins can exhibit strict subcellular localization and can be included in endocytic vesicles (Alberts B et al (1994) Molecular Biology of the Cell, Garland Publishing, Hamden Conn.).
Each G protein is composed of alpha (.alpha.), beta (.beta.) and gamma (.gamma.) subunits associated as a complex in the inactive, GDP-bound form. Activation of a transmembrane receptor by a hormone results in activation of the GTPase and replacement of GDP by GTP. The activated heterotrimer, the activated .alpha. subunit, or the .beta.-.gamma. subunit may have specific activity. Generally, the .alpha. subunit of a G protein disassociates from the .beta. and .gamma. complex, interacts with receptors and carries the message to the effector.
There are at least 20 genes for G.alpha. subunits which encode four major classes of proteins with at least 56-95% amino acid identity. The stimulatory, Gs class, is sensitive to pertussis toxin which uncouples the receptor:G protein interaction. This uncoupling blocks signal transduction to those receptors that decrease the cAMP which regulates ion channels and activates phospholipases. The inhibitory, Gi class, is also susceptible to modification by pertussis toxin which prevents Gi from lowering cAMP levels. Two novel classes refractory to pertussis toxin modification, are Gq which activates phospholipase C and G.sub.12 which has sequence homology with the Drosophila gene concertina which may contribute to the regulation of embryonic development. The G.alpha. subunits range in molecular weight from 39-52 kDa and include some splice variants. Multiple genes also encode at least four .beta. and six .gamma. subunits which range in molecular weight from 35-36 kDa and 6-10 kDa, respectively (Watson S and S Arkinstall (1994) The G protein Linked Receptor Facts Book, Academic Press, San Diego Calif.).
The .beta.-.gamma. dimer promotes the association of the GDP-bound .alpha. subunit with ligand-bound receptor. The dimer both orients and stabilizes the association so that signal transduction does not occur in the absence of agonist. Neer E J (1995; Cell 80:249-257) reported that .beta.-.gamma. dimers interact with adenylyl cyclase, phospholipase C .beta., calmodulin, .beta. adrenergic receptor kinase, phospholipase A2, phosducin, phosphoinositide 3-kinase, transducin, etc. In addition, the dimer may regulate potassium channels, mediate mitogen-activated protein kinase pathways and activate or increase phosphoinositide hydrolysis. In yeast, the dimer mediates a G protein-dependent mating response. The five .beta. subunit isotypes share 53-90% amino acid identity and are expressed ubiquitously although it must be noted that .beta.-4 is more abundant in brain and lung than in other tissues (Clapman D E and E J Neer (1993) Nature 365:403-6).
The known .gamma. subunits from bovine, rat and mouse tissues are most divergent in their N-terminal sequence. The .gamma. subunits generally display at least one cysteine residue in approximately the middle of their amino acid sequence (between residues 35 and 45) which is important for dimer formation, ie, the cysteine in the .gamma. subunit cross links with a cysteine in the .beta. subunit. Many of the sequences show a C-terminal consensus sequence CAAX (where A represents aliphatic residues and X is unspecified) which resembles the ras oncogene terminal sequence and is a site for post-translational modification. The modification involves cleavage of the 3' terminal residues and subsequent carboxymethylation, farnesylation, geranylgeranylation or isoprenylation. Post-translational modification increases subunit diversity and hydrophobicity and is important for membrane association and functional activity. In contrast, the rat .gamma.-5 sequence which terminates in CSFL is widely expressed and was highly expressed in kidney, heart, lung, and brain.
Although the different G proteins subunits could form some 600 different combinations, not all combinations are possible or functional. In the case of dimers, the .beta.1-.gamma.1 is only active in retina. Furthermore, the pattern of effector regulation may be highly specific. For example, whereas one type of adenylyl cyclase is activated by the G.alpha. subunit and unaffected by the .beta.-.gamma. subunit, a second type is activated by .alpha. subunit and inhibited by .beta.-.gamma. subunit. In another example involving the pituitary-derived GH3 cell line, the somatostatin receptor and the muscarinic receptor both regulate calcium channels, but each uses an alternatively spliced form of the .alpha..sub.s/o and different .beta.-.gamma. subunits. A final example addresses specificity and efficiency; in reconstituted vesicles, the .beta.-adrenergic receptor activates Gs as much as 3-fold better than Gi and the .beta.-.gamma. subunits from either heterotrimer should activate the potassium channel, however, only adenylyl cyclase is activated.
Neer (supra) suggests that G protein regulation depends on a combination of factors including the kinetics of ATP hydrolysis, stoichiometry, covalent modification, accessory proteins and compartmentalization, and that the number of receptors exceeds the number of G proteins. The molecular and functional diversity of Gs-stimulated adenylyl cyclases was recently reviewed by lyengar R (1993; FASEB Jour 7:768-75), and different tissues were shown to express a variety of adenylyl cyclases which were differentially regulated by the .beta.-.gamma. dimers and other molecules.
Diseases Associated with Cell Signaling Molecules and Pathways
Mutations in the molecules and alterations in the expression pattern of the components of the cell signaling cascade may result in abnormal activation of leukocytes or lymphocytes or cellular proliferation which affects growth and development. Inappropriate activation of leukocytes or lymphocytes may result in the tissue damage and destruction seen in autoimmune diseases such as rheumatoid arthritis, biliary cirrhosis, hemolytic anemia, lupus erythematosus, and thyroiditis. For example, Aussel C et al. (1988; J Immunol 140-215) reported that T cell activation is a G protein regulated process. Work in Jurkat cells with pertussis toxin showed that G protein serves as a transducer for signals via the T cell receptor-CD3 complex. In addition, the fact that fluoride ions stimulate the release of diacylglycerol but not inositol phosphate 3 further suggests that G proteins control the activity of phospholipase C.
Abnormal proliferation of cells can cause endometriosis or tumors, adenomas or carcinomas. Cyclic AMP stimulation of brain, thyroid, adrenal, and gonadal tissue proliferation is regulated by G proteins. In fact, about 50 percent of growth hormone-producing pituitary adenomas contain a mutated G.alpha..sub.S allele, and similar mutations have been associated with thyroid carcinomas and the neoplastic lesions of McCune-Albright syndrome. A known mutation in the G.alpha..sub.2i gene is found in tumors derived from adrenal cortex and ovary. Persistent extracellular stimulation and expression of those receptors coupled to Gq and phospholipase C can also result in tumor formation (Isselbacher K J et al (1994) Harrison's Principles of Internal Medicine, McGraw-Hill, New York N.Y.). In addition, multiple endocrine hyperfunction may be due to defects in the G protein-cyclic AMP-protein kinase A-dependent pathway.
Phosphoinositide 3 kinase is a key signaling enzyme implicated in receptor stimulated mitogenesis, oxidative bursting in neutrophils, membrane ruffling and glucose uptake. Stephens L et al. (1994; Cell 77:83-93) report that phosphoinositide 3 kinase activation in myeloid derived cells is regulated by .beta.-.gamma. dimers as well as phosphotyrosine kinase. Furthermore, it appears that tissue specificity may be governed by concentration of .beta.-.gamma. dimer molecules and that activation is more rapid and transient than that regulated by phosphotyrosine kinase. Although it was not suggested, it appears that the ability to control expression of either .beta. or .gamma. subunits provides a means to regulate cell signaling and mitogenesis.
The diversity of G subunit proteins, their functional combinations and their interactions with receptors present opportunities to intercede in abnormal cell processes. The activation of G proteins and the rate of GTP hydrolysis can be altered by controlling subunit production and association. Preventing dimer and heterotrimer formation can diminish cell signalling in GTP regulated pathways, reducing the activation of second messengers and controlling activation of leukocytes and lymphocytes and cell proliferation associated with endometriosis and tumor formation.